Cancer progression depends on the action of proteolytic systems that facilitate the growth and invasion of tumor cells. The membrane type-1 matrix metalloproteinase (MT1-MMP) endows tumor cells with the ability to invade and grow within collagenous matrices and thus is a key protease in cancer progression. As a membrane-tethered protease, MT1-MMP is regulated by a dynamic interplay of regulatory mechanisms that collectively control the level of active enzyme on the tumor cell surface and in the pericellular space. The long term objective of this application is to unveil the mechanisms regulating MT1-MMP activity at the tumor cell surface and apply this knowledge towards the development of new approaches aimed at inhibiting MT1-MMP in cancer. Our previous effort has been focused on elucidating the structural features and biochemical processes that define the ability of MT1-MMP to undergo autocatalytic processing and ectodomain shedding on the cell membrane, two fundamental processes of enzyme regulation. Processing of active MT1-MMP yields an inactive membrane-tethered fragment of 44 kDa that maintains key enzyme domains but its function in MT1-MMP regulation is poorly understood. Ectodomain shedding of MT1-MMP yields a 50-kDa soluble form that is present in tumors and is a fully competent protease. However its contribution to tumor proteolysis is unknown. Herein, new evidence in vitro and in tumor xenografts shows that the membrane-tethered 44-kDa species, displays a dynamic interaction with active MT1-MMP and identifies this fragment as a complex regulator of enzyme function in tumor cells. The naturally shed ectodomain of MT1-MMP has been characterized and found to be a catalytically competent protease, sensitive to TIMP-2, which has the potential to expand the proteolytic repertoire of MT1-MMP from the confines of the cell membrane to the pericellular space and regulate the activity of membrane-anchored MT1-MMP. Collectively, these observations pose a new paradigm in the regulation of MT1-MMP activity and suggest the hypothesis that processed and soluble forms of MT1-MMP play critical roles in tumor malignancy. To test this hypothesis we propose to: (1) investigate the dynamic interplay between the processed and active forms of MT1-MMP in enzyme function, (2) define the structural basis for the effects of the 44-kDa species on MT1-MMP regulation, (3) investigate the role of the soluble MT1-MMP in the regulation of MT1-MMP activity and (4) investigate the role of processed and soluble forms of MT1-MMP in functional assays of tumor cell invasion and growth in vitro and in vivo. The results of this application will contribute to our understanding of MT1-MMP function in tumor cells and contribute to the collective effort aimed at inhibiting its activity in cancerous tissues. [unreadable] [unreadable]